Project Abstract Synucleinopathies are a class of neurodegenerative disease characterized by pathological lesions composed of aggregates of alpha-synuclein (alpha-syn) in select populations of neurons. Alpha-synuclein is a small, abundant lipid-binding protein of unknown function that is prone to misfolding when not bound to lipids. The accumulation of alpha-syn aggregates in synucleinopathies suggests that aberrations in protein homeostasis might contribute to pathogenesis in these diseases. Because protein homeostasis mechanisms are highly conserved throughout eukaryotes, yeast cells can be used as a tractable model organism to investigate factors that contribute to the pathogenesis of synucleinopathies. The Lindquist lab has developed a yeast model over-expressing human alpha- syn that recapitulates many aspects of alpha-syn toxicity, and has observed that sterol biosynthesis and trafficking are also perturbed. Disrupting sterol biosynthesis can potentially alter a variety of cellular pathways. In fact, sphingolipid production is responsive to flux through the sterol pathway. Interestingly, both pathways have been implicated independently in alpha-syn toxicity and disease progression. The contribution of lipids to alpha-syn toxicity remains unresolved, and understanding how lipids affect synucleinopathy pathogenesis is essential for identifying potential therapeutic targets. The first aim of my proposal will investigate how the yeast lipidome is altered in response to alpha-syn expression. Lipidomic profiling has performed on yeast expressing nontoxic and toxic levels of alpha-syn in collaboration with Dr. Clary Clish at the Broad Institute. The lipid species identified up to this point correlate with those predicted to change upon alpha-syn expression. These results will then be compared to the lipid profiles of yeast co-expressing alpha-syn and genetic modifiers we discovered using overexpression and deletion screens. This will identify the lipids that change when toxicity is suppressed or enhanced. These targets lipids will then be manipulated in our yeast alpha-syn model to determine their effect on toxicity. My second aim is to evaluate whether the defects in cellular unesterified ergosterol distribution caused by alpha-syn expression lead to the mis-trafficking and accumulation of sphingolipids. Fluorescent microscopy will be used to monitor sphingolipid trafficking and localization in yeast cells. In addition, sphingolipids will be quantified using metabolic labeling. The cellular localization of unesterified ergosterol will be determined using filipin stain. Finally, my last aim will be evaluate sterol-sphingolipid regulation defects in cultured DA neurons expressing alpha-syn. Similar to aim 2, fluorescent microscopy will be used to monitor the trafficking and localization of unesterified cholesterol and sphingolipids, respectively. The amount of sphingolipids and sterols in alpha-syn expressing DA neurons will be quantified as well.